Current Pharmaceutical Design, 2007, 13, 2057-2073 2057

Heparanase: Structure, Biological Functions, and Inhibition by Heparin-Derived

Mimetics of Heparan Sulfate
Israel Vlodavsky1,*, Neta Ilan1, Annamaria Naggi2 and Benito Casu2,*

1
Cancer and Vascular and Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096 and 2G. Ron-
zoni Institute for Chemical and Biochemical Research, via G. Colombo, 81, 20133 Milan, Italy

Abstract: Heparanase is an endoglycosidase which cleaves heparan sulfate (HS) and hence participates in degradation and remodeling of
the extracellular matrix (ECM). Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an
invasive phenotype in experimental animals. The enzyme also releases angiogenic factors from the ECM and thereby induces an angio-
genic response in vivo. Heparanase upregulation correlates with increased tumor vascularity and poor postoperative survival of cancer
patients. Heparanase is synthesized as a 65 kDa inactive precursor that undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa pro-
tein subunits that heterodimerize to form an active enzyme. Heparanase exhibits also non-enzymatic activities, independent of its in-
volvement in ECM degradation. Among these, are the enhancement of Akt signaling, stimulation of PI3K- and p38-dependent endothe-
lial cell migration, and up regulation of VEGF, all contributing to its potent pro-angiogenic activity. Studies on relationships between
structure and heparanase inhibition activity of nonanticogulant heparins systematically differing in their O-sulfation patterns, degrees of
N-acetylation, and glycol-splitting of both pre-existing nonsulfated uronic acid residues (prevalently D-glucuronic) and/or those (L-
iduronic acid/L-galacturonic acid) generated by graded 2-O-desulfation, have permitted to select effective inhibitors of the enzymatic ac-
tivity of heparanase. N-acetylated, glycol-split heparins emerged as especially strong inhibitors of heparanase, exerting little or no release
of growth factors from ECM. N-acetylated glycol-split species of heparin, as well as heparanase gene silencing inhibit tumor metastasis,
angiogenesis and inflammation in experimental animal models. These observations and the unexpected identification of a single func-
tional heparanase, suggest that the enzyme is a promising target for anti-cancer and anti-inflammatory drug development.
Key Words: Heparanase, angiogenesis, heparanase inhibitors, heparin, heparin derivatives.

1. INTRODUCTION of modulating the activity of growth factors such as FGF-2 and

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macro-
molecules associated with the cell surface and ECM of a wide range
of cells of vertebrate and invertebrate tissues [1, 2]. The basic
HSPG structure consists of a protein core to which several linear
heparan sulfate (HS) chains are covalently O-linked. The polysac-
charide chains are typically composed of repeating hexuronic acid
and D-glucosamine disaccharide units that are modified at various
positions by sulfation, epimerization and N-acetylation, yielding
clusters of sulfated disaccharides separated by low or non-sulfated
regions [1-3]. Heparan sulfate (HS) glycosaminoglycans bind to
and assemble ECM proteins (i.e., laminin, fibronectin, collagen
type IV) and thereby contribute significantly to the ECM' self as-
sembly and integrity. HS also play important roles in cell-cell and
cell-ECM interactions. Moreover, the HS chains, unique in their
ability to bind a multitude of proteins, ensure that a wide variety of
bioactive molecules bind to the cell surface and ECM and thereby
function in the control of normal and pathological processes, among
which are morphogenesis, tissue repair, inflammation, vasculariza-
tion, and cancer metastasis [1-5]. Apart from sequestration of bioac-
tive molecules, transmembrane (syndecans) and phospholipid-
anchored (glypicans) HSPGs have a co-receptor role in which the
proteoglycan, in concert with the other cell surface molecules,
comprises a functional receptor complex that binds the ligand and
mediates its action [1-5]. HS also tether a multitude of growth fac-
tors, chemokines, cytokines and enzymes to the ECM and cell sur-
face, providing a low affinity storage depot for heparin-binding
proteins [4], including the potent pro-angiogenic factors FGF-2 and
VEGF [6-9]. Cleavage of HS side chains is therefore expected not
only to alter the integrity of the ECM, but also to release HS-bound
biological mediators. In addition, HS fragments are also capable
*Address correspondence to these authors at the Vascular and Tumor Biol-
ogy Research Center, Rappaport Faculty of Medicine, Technion, P.O. Box
9649, Haifa 31096, Israel; Tel: 972-4-8295410; Fax: 972-4-853947;
E-mail: Vlodavsk@cc.huji.ac.il
G. Ronzoni Institute for Chemical and Biochemical Research – via G. Co-
lombo, 81, Milan 20133, Italy; Tel: +39-02-7064-1623; Fax: +39-02- 7064-
1634; E-mail: casu@ronzoni.it
VEGF and enzymes such as thrombin and lipoprotein lipase. The
ECM provides an essential physical barrier between cells and tis-
sues, as well as a scaffold for cell growth, migration, differentiation
and survival, and undergoes continuous remodeling during devel-
opment and in certain pathological conditions such as wound heal-
ing and cancer [10]. ECM-remodeling enzymes are thus expected to
profoundly affect cell and tissue function. While intensive research
focused on enzymes capable of degrading and remodeling protein
components in the ECM [11,12], less attention was paid to enzymes
(e.g., heparanase) cleaving glycosaminoglycan side chains.
Heparanase is an endo-
-D-glucuronidase capable of cleaving
HS side chains at a limited number of intrachain sites, yielding HS
fragments of still appreciable size (~5-7 kDa) [13-18]. Heparanase
activity has long been detected in a number of cell types and tis-
sues. Importantly, heparanase activity correlated with the metastatic
potential of tumor-derived cells, attributed to enhanced cell dis-
semination as a consequence of HS cleavage and remodeling of the
ECM barrier [19, 20]. Similarly, heparanase activity is implicated
in neovascularization, inflammation and autoimmunity, involving
migration of vascular endothelial cells and activated cells of the
immune system [19-21]. In spite of the attractive clinical relevance
of the pro-metastatic, pro-inflammatory and pro-angiogenic activi-
ties of heparanase, progress in the field was slow, largely due to the
lack of recombinant heparanase, molecular probes, antibodies, and
a simple bioassay to quantify heparanase activity.
Heparanase activity was attributed to proteins with molecular
weight ranging from 8 to 130 kDa, raising the possible existence of
several HS-degrading endoglycosidic enzymes [19-21]. This confu-
sion was solved when the cloning of a single human heparanase
cDNA sequence was independently reported by several groups [22-
25]. With the availability of appropriate reagents, heparanase re-
search entered a new era. Recent studies have shown that hepara-
nase is up regulated in an increasing number of primary human
tumors [26]. Heparanase upregulation correlates with increased
lymph node and distant metastasis, increased micro-vessel density,
and reduced post-operation survival of cancer patients [19,20,26,
27], providing a strong clinical support for its pro-metastatic and
pro-angiogenic features and for heparanase as a promising target for

1381-6128/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

2058 Current Pharmaceutical Design, 2007, Vol. 13, No. 20
the development of anti-cancer treatments. Here, we summarize
recent progress in molecular and cellular aspects of heparanase, and
outline strategies for the development of heparanase inhibitors.
Taking into account the estabished role of HS in cancer [28,29] and
recent clinical trials demonstrating a beneficial effect of heparin in
cancer patients [30-33], we discuss relationships between structure
and heparanase inhibition activity of heparin derivatives, with spe-
cial emphasis on non-anticoagulant glycol-split species which spe-
cifically inhibit heparanase enzymatic activity and experimental
metastasis.
2. MOLECULAR AND BIOCHEMICAL PROPERTIES OF
HEPARANASE
2.1. Structure
Only one gene (HPSE) has been shown to encode for a protein
with heparanase activity [22-25]. Sequence analysis revealed that
heparanase is highly conserved, with similar sequences found in
human, rat, mouse, cow, chicken, molluscs and zebra fish [19, 34].
The human heparanase cDNA contains an open reading frame that
encodes a polypeptide of 543 amino acids with a molecular weight
of 61.2 kDa. The active heparanase purified from placenta, plate-
lets, and various cell lines was found to lack its N-terminal 156
amino acids, suggesting post-translational proteolysis of the
heparanase polypeptide [22-25]. In fact, active heparanase was
subsequently reported to be a heterodimer consisting of a 50 kDa
subunit (Lys158–Ile543) associated non-covalently with an 8 kDa
peptide (Gln36–Glu109). The intervening 6 kDa peptide (Ser110–
Gln157) is excised by proteolysis [35-37]. Based on the predicted
amino acid sequence, the 50 kDa subunit of human heparanase
contains six putative N-glycosylation sites. Although glycosylation
was not required for enzyme activity, secretion of heparanase was
regulated by glycosylation [38]. The sequence also contains a 35-
amino acid N-terminal signal sequence (Met1–Ala35) and a C-
terminal hydrophobic domain (Pro515-Ile534). Protein sequence
alignment approaches in combination with secondary structure
predictions indicated that heparanase contains sequences that are
homologous to families 10, 39, and 51 of the clan A glycosyl hy-
drolyses, especially in terms of the active-site regions [39]. This
clan of enzymes uses a general acid catalysis mechanism for the
hydrolysis of glycosidic bonds. The mechanism requires two criti-
cal residues, a proton donor and a nucleophile, both of which ap-
pear to be conserved in heparanase at Glu225 and Glu343, respec-
tively [39]. Site-directed mutagenesis of these residues completely
abolished heparanase activity, indicating that heparanase uses a
catalytic mechanism characteristic of clan A glycosyl hydrolases
[39].
2.2. Processing and Activation
Taking into account the multitude of polypeptides associated
with HS on the cell surface and ECM and their ability to profoundly
affect cell and tissue function, heparanase activity and bioavail-
ability should be kept tightly regulated. While regulation at the
transcriptional level [40-44] represents one type of control mecha-
nism, regulation at the post-translational level (i.e., heparanase
processing, cellular localization and secretion) provides an addi-
tional key regulatory mechanism. A major 50 kDa protein is de-
tected in cell lysates following transfection and over expression of
the heparanase cDNA, correlating with high levels of enzymatic
activity [25]. In contrast, a 65 kDa protein was found in the cell
conditioned medium, raising the possibility that the protein is first
synthesized as a latent proenzyme, which is then activated by pro-
teolytic processing. Purification of the human heparanase to homo-
geneity enabled N-terminus sequencing of the 50 kDa protein [35].
Interestingly, the purified heparanase preparation was noted to in-
clude an 8 kDa protein and further analysis revealed that this pro-
tein is derived from the N-terminus region (Gln36-Glu109) of
heparanase, immediately next to the signal sequence [35]. This
finding led the authors to suggest that active heparanase is a het-
Casu et al.
erodimer composed of the 8 and 50 kDa subunits. Indeed, attempts
to express the truncated 50 kDa (Lys158-Ile543) protein alone yielded
no enzymatic activity [39], indicating that an N-terminus sequence
is required. This hypothesis was confirmed by co-transfection and
immunoprecipitation approaches, convincingly demonstrating that
the two subunits are associated with each other, and that enzymatic
activity is only obtained by co-expression of both the 8 and 50 kDa
subunits [36,37]. Multiple sequence alignment and secondary struc-
ture prediction suggest that heparanase adopts a TIM barrel fold,
similar to other glycosyl hydrolases [39]. This fold motif usually
consists of eight alternating
-helices and -strands. Within the
heparanase 50 kDa subunit, clear homology was noted for only six

/ units, leading Nardella et al. to suggest that the two other units
are contributed by the 8 kDa subunit. Indeed, structural prediction
revealed the presence of a /
/ element in the 8 kDa subunit,
which may thus contribute the missing TIM barrel units [45]. These
and other [37] studies, indicated that the linker domain (Ser110-
Gln157) inhibits heparanase activity and needs to be fully removed.
Adopting site-directed mutagenesis to identify amino acids essential
for cleavage at Glu109-Ser110 (site 1) and Gln157-Lys158 (site 2), Ab-
boud-Jarrous et al. reported that none of the mutations generated at
site 1 and its flanking regions had an effect on heparanase process-
ing and activity [46]. In contrast, substitution of Tyr156 (site 2) by
alanine or glutamine rendered heparanase inactive and improperly
processed [46]. Subsequent studies revealed that a bulky hydropho-
bic amino acid (i.e., Tyr156) at position 2 (P2) of the cleavage site
(Gln157-Lys158) is absolutely required for heparanase processing and
activation, resembling the cleavage specificity of cathepsin L [46].
Indeed, incubation of purified latent 65 kDa heparanase with
cathepsin L yielded properly processed and active heparanase,
composed of the 50 and 8 kDa subunits. Processing and activation
of the pro-enzyme by intact cells and in a cell free system was in-
hibited in the presence of a specific, cell permeable inhibitor of
cathepsin L [46]. Applying a structural model, we demonstrated
that the linker segment, or even a small 1 kDa portion at its C-
terminus, render the active site inaccessible to the HS substrate.
Moreover, heparanase activity was inhibited in the presence of a 1
kDa peptide corresponding to the C-terminus of the linker segment.
While predicted model structures do provide important information
(Fig. 1), it is limited by the relatively low sequence homology with
other glycosyl hydrolases, and awaits further confirmation by crys-
tallization and x-ray analysis. Site directed mutagenesis of aromatic
residues along the linker segment, supports a mechanism by which
cathepsin L is not only responsible for cleavage at site 2, but is
actually capable of a stepwise cleavage and removal of the entire
linker region, suggesting that processing and activation of pro-
heparanase can be brought about solely by cathepsin L and/or
cathepsin L-like proteases (our unpublished results). It is likely that
cathepsins other than cathepsin L may activate pro-heparanase,
possibly in a cell- and tissue- dependent manner.
Recent findings indicated that heparanase processing occurs in-
tracellularly [47-49], pointing to acidic vesicles, most likely
lysosomes, as the processing organelles. Following exogenous addi-
tion, heparanase was noted to reside within perinuclear endocytic
vesicles identified as late endosomes [50] and lysosomes [51]. Ap-
plying anti-heparanase antibodies that distinguish between the la-
tent and processed heparanase forms, we have demonstrated that
not only the processed, but also the 65 kDa latent heparanase was
localized in endocytic vesicles, indicating that processing does not
take place at the cell surface [49]. Complete inhibition of hepara-
nase processing by chloroquine and bafilomycin A1, reagents that
raise the pH of acidic vesicles and thus inhibit the enzymatic activ-
ity of resident enzymes, further points to acidic vesicles as the
heparanase processing site [49]. Taking this notion a step further,
Cohen et al. utilized a cell fractionation approach and demonstrated
that lysosomal/endosomal, but not cytoplasmic preparation is capa-
ble of heparanase processing, yielding an active enzyme [52].
Moreover, processing by the lysosomal/endosomal preparation was
Heparanase Functions and Inhibition Current Pharmaceutical Design, 2007, Vol. 13, No. 20 2059

Formula
CH2OSO3- CO2- CH2OSO3-
O O O
OH O OH(SO3)
1 (AGA) O OH O

NHSO3- OH NHSO3-
(Ac)

CH2OSO3- CH2OSO3- COO-

O O O O
2 (H) OH
CO2- O OH OH
O
OH O
O
n>>m
NHSO3- OSO3- n NHAc OH m
(OH)

GlcNSO36SO3 - IdoA2SO3 GlcNAc6SO3 - GlcA

CH2OSO3- CH2OSO3- COO-

O O O O
CO2- O O
3 (NAH) OH
OH
OH
O
OH
O
n>>m NHAc OSO3- n NHAc OH m
(OH)

GlcNAc6SO3 - IdoA2SO3 GlcNAc - GlcA

CH2OSO3- CH2OSO3-
O O O O
50 CO2- COO- O
4 ( H gs ) OSO3-
OH
O OH
O
O

NHR OSO3- NHR OH OH n

CH2OSO3- CO2- CH2OSO3- CH2OSO3-

O O O O O
CO2-
5 (AGA*IA) OH
OH
O OSO3-
OH
O OH
O O O

NHAc OH NHSO3- OSO3- NHSO3-

CH2OSO3- CO2- CH2OSO3- CH2OSO3-

O O O O O
CO2-
OH O OSO3- O OH
6 (gs-AGA*IA) O O OH O

NHAc OH OH NHSO3- OSO3- NHSO3-

CH2OSO3- CH2OSO3- CO2-

O O O O O
CO - CO2-
7 aza-oligoH OH2
O
OH
O OH
O OH
O OH
NR

OSO3- OSO3 OSO3-

OSO3- OSO3-

CH2OSO3- CH2OSO3- CH2OSO3- OSO3- CH2OSO3- CH2OSO3-

O O O O O O
8 MHS OSO3- OSO3- OSO3- OSO3- OSO3- OSO3-
O O O OSO3- O O
OSO3-
OSO3- OSO3- OSO3- OSO3- OSO3- OSO3-
2060 Current Pharmaceutical Design, 2007, Vol. 13, No. 20 Casu et al.

Fig. (1). Primary structure, critical amino acids and predicted three-dimensional structure of the heparanase heterodimer. Pre-proheparanase harbors 35 amino
acids signal peptide (s.p., Met1 -Ala35) which is removed upon entering the ER. The protein is then subjected to glycosylated at six N-glycosylation sites ( )
and secreted as a latent ~65 kDa protein (upper panel). Proteolytic processing removes the linker domain (Ser110–Gln157), resulting in an 8 kDa (Gln36–Glu109)
and 50 kDa (Lys158–Ile543) protein subunits (middle panel) that heterodimerize to yield an active enzyme. A predicted three-dimensional structure of the hepara-
nase heterodimer (bottom panel was created based on homology with 1,4--xylanase. Shown (left) are the 8 (yellow) and 50 kDa (gray) subunits and glutamic
acid residues 225 and 343 that comprise the enzyme active site (red). The heparin binding domains (HBD 1 and 2, blue and green) are in close proximity to the
enzyme active site (red). [See text.]

most efficient at acidic pH conditions (pH 4-5) [52], typical of the
lysosomal compartment. These results and the ability of cathepsin
L, a characteristic lysosomal enzyme, to properly process and acti-
vate pro-heparanase, strongly support the lysosomal compartment
as the site of processing.
2.3. Secretion
In spite of its localization to a highly active protein degradation
environment such as the lysosome, heparanase exhibits a half life of
about 30 hours [47], relatively long compared with a t1/2 of 2-6
hours, and 25 minutes of transmembrane and GPI-anchored
HSPGs, respectively. Residence and accumulation of heparanase in
late endosomes and lysosomes may indicate that the enzyme func-
tions in physiological turnover of cellular HSPGs [53]. Being the
enzyme not readily accessible to its extracellular substrate, this
suggests the existence of regulatory mechanism(s) by which intra-
cellular, lysosomal heparanase is secreted in response to local or
systemic cues. Recent observations may support the occurrence of
such a scenario. For example, treatment of human microvascular
endothelial cells (EC) with the pro-inflammatory cytokines TNF

and IL-1 resulted in a marked increase of heparanase secretion
[54]. Secretion of heparanase in response to TNF
was also noted
in human peripheral T-cells [55]. Interestingly, TNF
and IL-1
had no effect on heparanase secretion from tumor-derived cells (our
unpublished results), suggesting that effective stimuli may vary
among cell types and biological settings. Our recent studies indicate
that nucleotides, such as ATP, ADP and adenosine are highly effec-
tive in stimulating secretion of both active heparanase and cathepsin
D by tumor cells via activation of the PKC and PKA pathways
[Shafat et al. J Biol Chem 2006; 281: 23804-11].
2.4. Cellular Uptake and Extracellular Retention
Apart from storage in the endosomal/lysosomal compartment,
efficient uptake of exogenous heparanase by primary fibroblasts
and EC, as well as by tumor-derived cells [47, 49, 50] provides an
additional mechanism that limits retention of the enzyme extracel-
lularly. Several lines of evidence indicate that heparanase uptake is
mediated by cell surface HS. We have demonstrated that addition of
heparin or xylosides results in accumulation of heparanase in the
culture medium of heparanase transfected cells. Heparanase uptake
was attenuated in HS-deficient cells and in cells that were treated
with bacterial heparinase, but not with chondroitinase ABC. In
addition, transfection and over expression of heparanase in HS-
deficient cells resulted in accumulation of the latent pro-enzyme in
the culture medium, concomitant with decreased levels of the intra-
cellular processed enzyme [47]. This result suggests that intracellu-
Heparanase Functions and Inhibition
lar accumulation of processed heparanase occurs following uptake
of the secreted latent protein [47] (Fig. 2). Sequence alignment of
heparin binding domains (HBD) in the heparanase molecule re-
vealed the existence of two domains that match consensus se-
quences for heparin binding. These were mapped at Lys158-Asp162
at the N-terminus of the 50 kDa heparanase subunit, and at Pro271-
Met278 [56]. A peptide containing the Lys158-Asp162 sequence
(KKDC) exhibited firm binding to heparin and HS, and inhibited
both heparanase uptake and enzymatic activity, most likely due to
competition with the HS substrate [56]. Furthermore, heparanase
deletion mutants lacking each of the heparin binding domains ex-
hibited no enzymatic activity. Deletion of the KKDC sequence
(65
15) resulted in intracellular accumulation of the 65 kDa pro-
enzyme that failed to get secreted. Deletion of the Pro271-Met278
sequence (65
10) led to accumulation of the pro-enzyme in the cell
conditioned medium [56], further supporting a critical role for HS
in heparanase uptake and processing. 2D NMR analysis revealed a
strong interaction of the two putative HS binding regions of
heparanase with a heparin/HS pentasaccharide (i.e., AGA*IA, for-
mula 5) [56]. Notably, the KKDC sequence, and specifically ly-
sine158 and lysine159 involved in interaction of the enzyme with
heparin/HS [56], appeared to reside in close proximity to Glu225 and
Glu343, comprising the enzyme' active site in a micro pocket domain
[39] (Fig. 1). The predicted 3D model further emphasizes the micro
pocket region and, in particular, the KKDC sequence (Fig. 1) as a
valid target for the development of heparanase inhibiting molecules.
More recently, Vreys et al. have identified two additional cell sur-
face receptors that mediate heparanase uptake, namely the low den-
sity lipoprotein receptor-related protein (LRP) and the mannose 6-
phosphate receptor [48]. The precise contribution of each receptor
species to heparanase uptake is yet to be demonstrated. Collec-
tively, the above described studies emphasize the complexity and
tight regulation of heparanase expression, processing and secretion
(Fig. 2) supporting its potency and significance in normal and
pathological conditions.
3. HEPARANASE AND CANCER PROGRESSION
3.1. Pro-Angiogenic Properties
HSPGs are prominent components of blood vessels, and HSPG
degrading enzymes have long been implicated in a number of angi-
ogenesis-related cellular processes. A critical early event in the
angiogenic process is degradation of the subendothelial basement
membrane (BM), followed by endothelial cell (EC) migration to-
ward the angiogenic stimulus. Similar to its involvement in tumor
cell dissemination, it is conceivable that by degrading HS in the
BM, heparanase may directly facilitate EC invasion and sprouting.
Indeed, heparanase expression by FGF-2 stimulated bone marrow-
derived EC was demonstrated by RT-PCR [57]. Immunohisto-
chemistry of tumor specimens revealed heparanase staining of EC
in capillaries, but not mature blood vessels [57,58]. Moreover, by
releasing HS-bound angiogenic growth factors (i.e., FGF-2, VEGF)
from the ECM [7] active heparanase may indirectly facilitate EC
migration and proliferation [59]. In fact, given the multitude of
biological mediators that are sequestered by HS in the ECM [60],
heparanase activity liberates a number of active molecules that may
act cooperatively or synergistically to promote neovascularization.
An idealized picture of heparanase-induced release and activation
of FGF-2 molecules originally sequestered by HS chains of HSPGs
is shown in Fig. (3).
Wound healing orchestrates multiple cell types (i.e., neutro-
phils, macrophages, fibroblasts, keratinocytes, endothelial cells),
soluble (i.e., growth factors, cytokines, chemokines) and insoluble
(ECM components) mediators in a complex sequence of events.
Orchestration and regulation of the rapidly developing new tissue
observed in wound healing depend not only on cells and bioactive
polypeptides, but also on the ECM microenvironment, and require
new blood vessel formation to nourish the newly formed granula-
tion tissue. Elevated heparanase expression was observed in the
Current Pharmaceutical Design, 2007, Vol. 13, No. 20 2061
wound granulation tissue and blood vessels [61]. Heparanase con-
tribution to wound healing and wound angiogenesis has been dem-
onstrated in several experimental settings. Increased amounts of
heparanase were found in the wound fluid of heparanase trans-genic
(hpa-tg) vs. control mice [61], in agreement with heparanase ex-
pression in healing wounds. Moreover, elevated heparanase levels
in the wound fluid correlated with a comparable elevation of FGF-2
[61], providing an in vivo support for the ability of heparanase to
release heparin-binding pro-angiogenic factors. Enhanced and per-
sistent wound angiogenesis was further demonstrated in the hpa-tg
mice by applying magnetic resonance imaging (MRI) [61]. This
effect was inhibited by a non-anticoagulant glycol-split heparin
(ST1514, corresponding to H50gs in Ref. [62], formula 4), endowed
with a heparanase inhibitory activity at nM concentrations [62] (see
Section 4.2), thus clearly supporting a role for heparanase in wound
angiogenesis [61]. In a rat/flap punch model, topical application of
highly active recombinant heparanase improved wound healing by
40% and enhanced wound angiogenesis [61]. Measurements taken
in the area of flap incisions revealed a significant increase in epithe-
lium thickness, suggesting that heparanase promotes keratinocyte
proliferation due to an improved bioavailability of factors such as
KGF and HB-EGF. In addition, immunostaining of wound sections
with anti-smooth muscle actin (SMA) antibody revealed a seven
fold increase in SMA-positive blood vessels in response to hepara-
nase treatment [61]. Thus, heparanase accelerates wound healing by
enhanced migration and proliferation of keratinocytes and stimula-
tion of wound blood vessel formation and maturation. The coordi-
nated, simultaneous release of a combination of HS-bound growth
factors (i.e., FGF-2, VEGF, HB-EGF, KGF) is unique to hepara-
nase and may account for its efficient neovascularization and
wound healing promoting effect. It should be noted that wound
healing is only one example for the involvement of heparanase in
tissue remodeling and neovasculari-zation. For example, hepara-
nase transgenic mice (hpa-tg) exhibit excess branching and widen-
ing of mammary gland ducts, and an accelerated rate of hair
growth, both accompanied by enhanced vascularization [63, 64].
3.2. Non-Enzymatic Functions Associated with Angiogenesis,
Cell Survival and Migration
Akt Activation
Heparanase over expression in human U87 glioma [65], HT 29
colon carcinoma [66], CAG myloma [67], MCF7 [68], MDA-MB-
231 [69], and MDA-MB-435 [70] breast carcinoma cells correlated
with enhanced xenograft tumor growth and vascularization. Using
the RIP-Tag2 tumor model, Joyce et al. have recently demonstrated
elevated levels of heparanase mRNA and protein upon the transi-
tion from normal to angiogenic islets, which further increased when
solid tumors were detected [71]. These studies support the notion
that heparanase not only facilitates tumor metastasis, but also con-
tributes to the angiogenic switch and subsequent growth of the pri-
mary tumor. Enhanced tumor progression correlated with elevation
in blood vessel density, revealed by staining with anti-PECAM-1
antibodies, as well as by MRI analysis [68]. At the molecular level,
heparanase over expression was noted to facilitate cell adhesion and
migration of tumor cells, primary EC and T lymphocytes, mediated,
al least in part, by 1-integrin and Rac activation [55, 65, 72].
Heparanase over expression in U87 glioma, as well as in several
other tumor derived cells, correlated with enhanced serine/threonine
protein kinase Akt/PKB phosphorylation levels [65]. Moreover,
exogenous addition of heparanase to primary EC markedly stimu-
lated Akt phosphorylation [73]. This effect occurred both in the
presence or absence of HS on the cell surface, was independent of
heparanase enzymatic activity, and was augmented by heparin [73].
At the cellular level, heparanase addition stimulated PI 3-kinase-
dependent EC migration and invasion, and significantly improved
EC rearrangement into lumen containing tube-like structures [73].
These observations imply that heparanase is capable of eliciting
angiogenic responses by a direct effect on EC. The ability of
2062 Current Pharmaceutical Design, 2007, Vol. 13, No. 20 Casu et al.

Fig. (2). A schematic presentation of a proposed model for heparanase biosynthesis, processing and trafficking. Pre-pro-heparanase is first targeted to the ER
lumen via its own signal peptide (Met1-Ala35, 1). The 65 kDa pro-heparanase is then shuttled to the Golgi apparatus, and is subsequently secreted via vesicles
that bud from the Golgi (2), a step that is specifically inhibited by Brefeldin A (BFA) [50]. Once secreted, heparanase rapidly interacts with cell membrane
HSPGs such as syndecan-family members (3) [47], mannose-6 phosphate receptor or LRP [48], followed by a rapid endocytosis of the heparanase-HSPG
complex (4) that appears to accumulate in endosomes [50]. This step is inhibited by cytochalasin D (Cyto. D) [50], heparinase [47], or heparin [47]. Conver-
sion of endosomes to lysosomes results in heparanase processing and activation (5) that, in turn, participates in the turnover of HS side chains in the lysosome.
Heparanase processing and activation is specifically inhibited by chloroquine and Bafilomycin A, inhibitors of lysosomal proteinases. Typically, heparanase
appears at perinuclear lysosomal vesicles (6). Such a trafficking route may be bypassed by several potential ways, such as direct conversion of exocytosed
vesicles to endosomes (dashed arrow). Lysosomal heparanase may translocate to the nucleus, where it affects gene transcription, thus contributing to a more
differentiated state of carcinoma cells [120, 121] (7), or can get secreted in response to local or systemic cues. Secretion of active heparanase heterodimer is
inhibited by PKC inhibitors (Bis), and P2Y receptor antagonists (MRS, PPADS) (8). The latent secreted heparanase can also interact with heparanase-binding
protein (HBP) and activate signaling components such as Akt, p38, Src, Pyk2 and integrins, leading to enhanced cell adhesion, migration, VEGF induction and
angiogenesis [47, 49, 55, 65, 72 ] (9).

heparanase to stimulate Akt suggests that heparanase may protect
tumor cells from apoptosis [68], although the survival promoting
mechanism has not been sufficiently elucidated. The ability of ex-
ogenously added heparanase to activate signal transduction cas-
cades, and its augmentation by heparin may indicate the existence
of a cell surface heparanase receptor (Fig. 2, HBP), possibly low
density lipoprotein receptor-related proteins (LRP) [48], yet this
aspect awaits further research and proper confirmation.
VEGF up Regulation
In addition to the above described pro-angiogenic effects attrib-
uted to heparanase enzymatic and non-enzymatic activities, hepara-
nase is also closely involved in VEGF gene regulation. Transfection
and over expression of heparanase in rat C6 glioma, MDA-MB-435
human breast carcinoma and human embryonic kidney HEK293
cells, were accompanied by a 3-6 fold increase in VEGF mRNA
and protein levels, correlated with enhanced Matrigel and tumor
xenograft vascularization [70]. Moreover, transfection of the highly
metastatic mouse B16-BL6 melanoma cells with heparanase-
specific siRNA resulted in a 75% decrease in heparanase mRNA
and VEGF levels. This implies that endogenous heparanase is inti-
mately involved in VEGF gene regulation. Interestingly, VEGF
elevation by heparanase correlated with increased p38 phosphoryla-
tion levels, a signaling pathway implicated in VEGF induction [70].
Nonetheless, p38 inhibitors had no effect on heparanase-mediated
VEGF up regulation, suggesting the operation of another signaling
pathway(s) elicited by heparanase. Screening of several additional
inhibitors led to the identification of Src, a kinase that was shown to
modulate VEGF transcription [74-76], as a mediator of VEGF up
regulation by heparanase. Moreover, Src inhibitors prevented
VEGF induction by heparanase and significantly attenuated cell
migration enhanced by heparanase, positioning Src as a critical
down stream component that mediates heparanase functions [70].
VEGF induction and Src activation require heparanase secretion
that was recapitulated by exogenous addition of the enzyme. Impor-
tantly, Src activation was noted also upon exogenous addition of
point mutated (Glu225, Glu343) heparanase that lacks enzymatic ac-
tivity. Thus, heparanase exerts enzymatic activity-dependent (i.e.,
release of FGF-2) and independent (i.e., VEGF induction) pro-
angiogenic effects. It therefore appears that in addition to its pro-
metastatic function (see below), heparanase affects several key
Heparanase Functions and Inhibition
Fig. (3). Idealized representation of cleavage of HS chains of a heparan sulfate proteoglycan (HSPG) by heparanase, with release of growth factors (GF), their
activation by dimerization favored by HS fragments, and formation of signaling ternary complexes with growth factor receptors (GFR) on the surface of endo-
thelial cells.
components in tumor progression, resulting in increased blood ves-
sel density and maturation, enhanced tumor cell motility, and acti-
vation of signaling mediators that govern tumor cell proliferation
(i.e., Src) and survival (Akt). Collectively, these effects position
heparanase as an attractive target for the development of anti cancer
drugs.
3.3. Pro-Metastatic Properties
Heparanase activity correlated with the metastatic potential of
mouse B16 melanoma [77,78] and Eb lymphoma [79] cells. Thus,
sub lines with higher potential for metastasis and organ colonization
exhibited a higher enzymatic activity than low- or non- metastatic
cells. These early observations gained substantial support when
specific molecular probes became available shortly after cloning the
heparanase gene. Hulett et al. employed Northern blot analysis to
study heparanase expression in cells and tissues [22], while Vlo-
davsky et al. utilized a transfection approach [25]. In these studies,
highly metastatic rat mammary adenocarcinoma cell lines were
noted to express high levels of heparanase mRNA transcripts com-
pared with their non metastatic counterpart cells [22]. Subcutaneous
inoculation of non metastatic Eb lymphoma cells engineered to over
express heparanase (hpa-Eb) resulted in a significant decease in
survival time of the mice due to a massive liver infiltration [25],
further supporting the correlation between heparanase expression
and the metastatic capacity of cancer cells.
We employed siRNA and ribozyme technologies to reduce
heparanase expression levels in a specific manner. Transfection and
stable expression of anti-heparanase ribozyme construct in human
MDA-MB-435 breast carcinoma cells, known to express high levels
of heparanase activity, or in Eb mouse lymphoma cells engineered
to over express the human heparanase gene (hpa-Eb), resulted in a
marked decrease in heparanase levels evaluated by RT-PCR and
heparanase enzymatic activity. This decrease correlated with 55 -
65% reduction in cellular invasion through a reconstituted basement
membrane (Matrigel) [80]. Moreover, mice inoculated (s.c) with
hpa-Eb lymphoma cells transfected with anti-heparanase ribozyme
exhibited a marked decrease in liver metastasis and survived sig-
nificantly longer than mice inoculated with cells transfected with
Current Pharmaceutical Design, 2007, Vol. 13, No. 20 2063
control ribozyme. Similarly, lung colonization of B16-BL6 mela-
noma cells was markedly (> 90%) reduced applying cells trans-
fected with anti-heparanase siRNA due to a marked inhibition of
both heparanase gene expression and enzymatic activity, naturally
expressed by these cells [80]. Subcutaneous primary tumors pro-
duced by hpa-Eb cells expressing the anti-heparanase ribozyme
were less vascularized, supporting the pro-angiogenic function of
the enzyme. Altogether, both over-expression and silencing of the
heparanase gene clearly indicate that heparanase not only enhances
cell dissemination, but also promotes the establishment of a vascu-
lar network that accelerates primary tumor growth and provides a
gateway for invading metastatic cells.
While the studies described above provide a proof-of-concept
for the pro-metastatic and pro-angiogenic capacity of heparanase,
the clinical significance of the enzyme in tumor progression
emerges from a systematic evaluation of heparanase expression in
primary human tumors. Immunohistochemistry, in situ hybridi-
zation, RT-PCR and real time-PCR analyses revealed that hepara-
nase is up regulated in essentially all human tumors examined [26,
81, 82]. Heparanase up regulation in primary human tumors corre-
lated in some cases with tumors larger in size and with enhanced
micro vessel density, as well as with reduced post-operative sur-
vival of cancer patients providing a clinical support for the pro-
angiogenic function of the enzyme [19,20,26,27].
4. INHIBITION OF HEPARANASE
4.1. Heparanase Inhibition Strategies
Attempts to inhibit heparanase enzymatic activity were initiated
already at the early days of heparanase research, in parallel with the
emerging clinical relevance of this activity. More recently, with the
availability of recombinant heparanase and the establishment of
high-throughput screening methods, a variety of inhibitory mole-
cules have been developed, including neutralizing antibodies, pep-
tides, small molecules, modified non-anticoagulant species of hepa-
rin, as well as several other polyanionic molecules, such as lamina-
ran sulfate, suramin and PI-88 [26,27]. Heparanase can be inhibited
either at the level of the pro-enzyme by preventing its intracellular
2064 Current Pharmaceutical Design, 2007, Vol. 13, No. 20
processing and activation, or the active enzyme, mostly extracellu-
larly. Heparanase inhibitors may be targeted to the enzyme active
site, as well as to one or both of its identified heparin/HS binding
regions. Although the present review is especially focused on hepa-
rin-like, HS mimics, a brief account of alternative reported hepara-
nase-inhibitory strategies is given.
Peptides Competing with the Heparin/HS Binding Domains of
Heparanase
As mentioned in Section 2.4, three potential heparin-binding
domains in the heparanase molecule were identified and evidence
was provided that one of these domains is mapped at the N-
terminus of the 50 kDa heparanase subunit [56]. A peptide corre-
sponding to this region (KKDC peptide: Lys 158 -Asn 171) physi-
cally associates with heparin and HS and inhibits heparanase enzy-
matic activity in a dose-responsive manner. Furthermore, antibodies
directed to this region inhibited heparanase activity and a deletion
construct lacking this domain exhibited no enzymatic activity [56].
We have introduced a cysteine residue at the peptide C-terminus
and found that oxygenation and spontaneous dimeri-zation of the
KKDC peptide significantly improved its heparanase-inhibiting
activity.
Studies on processing and activation of the 65 kDa heparanase
proenzyme revealed that the linker peptide must be totally removed
in order to enable interaction between heparanase and its substrate
[45, and our unpublished results]. As mentioned in section 2.2, a 1
kDa peptide corresponding to the C-terminus of the linker segment
was found to inhibit heparanase enzymatic activity, most likely by
hindering accessibility of the HS substrate to the enzyme' active
site.
Small Molecules Targeting at the Catalytic Domain
Non carbohydrate, small molecule heparanase inhibitors, exem-
plified by 1H-isoindole-5-carboxylic acid and benzoxazol-5-yl-
acetic acid, were reported. A novel class of 1-[4-(1H-benzo-imida-
zol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-ureas were
recently described as potent inhibitors of heparanase [27]. Among
these, compound 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea
displayed a high heparanase inhibitory activity (IC50 - 0.075KM) in
vitro and a good efficacy in a B16 experimental metastasis model
[83].
Monoclonal Antibodies and Microbial Metabolites
An attractive approach for the inhibition of heparanase is the
development and use of neutralizing monoclonal antibodies to the
protein. A monoclonal antibody has been reported which effectively
abolishes the activity of recombinant heparanase when used at a
protein : antibody ratio of 1:10 [27]. A neutralizing polyclonal anti-
body directed against a peptide corresponding to the heparanase
active site (residues G215 – D234) effectively inhibited restenosis
in a rat model [84]. Random, high-throughput screening of chemi-
cal libraries and microbial metabolites, and rational design of com-
pounds that block the heparanase active site or ligand-binding do-
main are among the other approaches applied to develop effective
heparanase inhibitors [26,27,83]. Natural endogenous heparanase
inhibitors may also be identified. Further defining the heparanase
substrate specificity, catalytic and non-catalytic activities, as well as
the enzyme X-ray crystal structure is needed for pursuing a more
‘rational’ approach to develop effective and highly specific hepara-
nase inhibiting molecules.
In an attempt to apply gene silencing for therapeutic purposes, a
reliable system for siRNA administration, applying in vivo electro-
poration of siRNA expressing vectors, or lentiviral infection, was
recently developed. The effectiveness of this approach for suppress-
ing the biological activity of heparanase was judged by the inhibi-
tory effect of siRNA administration in several experimental models
of heparanase-driven biological processes, such as delayed type
hypersensitivity (DTH) inflammation [85], hair growth, and pros-
tate tumor progression (Vlodavsky et al., unpublished results).
Casu et al.
4.2. Heparin and Heparin Derivatives as Mimetics of Heparan
sulfate and Heparanase Inhibitors
As mentioned in Section 2, the active form of heparanase is a
50 kD protein containing a site responsible for cleavage of glycosi-
dic bonds of
-D-glucuronic acid (GlcA) residues of HS chains.
The minimum HS motif recognized and cleaved by the the enzyme
is trisaccharide 1, where the arrow indicates the site of cleavage.
The GlcA residue is flanked by two 6O-,N-sulfated glucosamine
residues, the first of which could alternatively be N-acetylated and
the second one also 3-O-sulfated [14,16]. Cleavage by heparanase
can be observed when sequence 1 is part of pentasaccharides [A.
Bisio, J.-P. Li, et al., unpublished; M. Petitou, personal communica-
tion).
As also described in Section 2, heparanase (both the latent and
active forms) contains three basic clusters, two of which may be
involved in tight binding of HS [56]. Widely pursued strategies for
inhibiting heparanase aim at preventing binding of HS chains to the
enzyme. Among other strategies (see Section 4.1), this goal could
be achieved using HS mimics that compete with HSPGs for the
basic clusters of heparanase involved in binding and recognition of
HS chains. Two chemically fully sulfated oligosaccharides (malto-
hexaose sulfate, MHS, and phosphomannopentaose sulfate, PI-88),
were shown to be good heparanase inhibitors [27,86]. Being not
close mimics of the natural substrate of heparanase, MHS and PI-88
are not covered in detail in the present review.
Heparin, an animal polysaccharide belonging to the glycosa-
minoglycan (GAG) family, is one of the closest mimics of HS [3]
and is a natural choice as heparanase inhibitor. Indeed, heparin has
been shown to be a potent inhibitor of the enzyme [87, 88]. How-
ever, the strong anticoagulant activity of this GAG, mainly associ-
ated with specific pentasaccharide sequences (AGA*IA, formula 5)
that constitute the active site for antithrombin (AT) [3], makes
heparin unsuitable for long-term treatments. As represented in For-
mula 2, heparin is prevalently made up by regular disaccharide
sequences of L-iduronic acid 2-sulfate and N-and 6-O-sulfated
glucosamine, but it is also constituted by undersulfated, GlcA- and
GlcNAc-containing sequences, mostly arranged in blocks [3, 89].
Heparin accordingly contains also some of sequences 1 recognized
and cleaved by heparanase [14, 16] hence acting as a substrate for
the enzyme, with behavior as inhibitor conceivably depending on
content and molecular environment of cleavable sequences. An-
other undesired property of heparin is its capability to displace
growth factors sequestered by HS chains in the ECM and on cell
surfaces [87]. When complexed with heparin, these growth factors
are activated and form with receptors of growth factors (GFR) ter-
nary complexes that trigger mitogenic signals, as illustrated in Fig.
(3) for HS fragments released by heparanase [4]. Ideal hepar-
anase inhibitors should accordingly be nonanticoagulant heparin
species endowed with potent antiheparanase activity without favor-
ing release and activation of growth factors.
Early screening of heparin derivatives permitted to identify
some structural features of the polysaccharide associated with inhi-
bition of heparanase. No significant differences were found be-
tween the currently used unfractionated heparins, low-molecular
weight heparins (LMWH) and a tetradecasaccharide heparin frag-
ment; however, a heparin hexasaccharide is a poor inhibitor of the
enzyme. Typical heparanase-inhibition curves, showing the gel
filtration profiles of sulfate labeled degradation fragments released
by heparanase from metabolically labeled ECM in the absence
(control) and presence of 5 g/ml and 0.25 g/ml of various hepa-
rin species are presented in Figs. (4A and B), respectively. Inhibi-
tion is reflected by the decreased amounts and Kav values of HS
fragments released from ECM and eluted as fractions 20-35, in
comparison with control incubation of the ECM with recombinant
heparanase in the absence of inhibitors [62, 88, 90]. Heparanase
activity is calculated as the total amount of cpm eluted in peak cor-
responding to fractions 20-35 multiplied by the Kav (i.e., elution
position) of these fragments.
Heparanase Functions and Inhibition
Fig. (4). Representative heparanase-inhibition curves for heparins and various heparin species. [A: from Ref. 91; B: from Ref. 62]. Sulfate labeled ECM was
incubated (4 h, 37°C, pH 6.0) with recombinant human heparanase (40 ng/ml) in the absence (control) and presence of 5 g/ml (A) or 0.25 g/ml (B) heparin
or heparin derivatives. Sulfate labeled degradation fragments relesed into the incubation medum were analyzed by gel filtration on Sepharose 6B. The peak
corresponding to fractions 20-35 was used to calculate the percent residual activity of the enzyme.
As illustrated in Fig. (4A), the heparanase inhibiting activity of
heparin derivatives increases with increasing degrees of sulfation.
However, N-sulfate groups seem not essential, since their replace-
ment by N-acyl groups (N-acetyl, N-succinyl, or N-hexanoyl)
yielded products still retaining significant inhibitory activity
[62,91]. (The structure of fully N-acetylated heparin (NA-H) is
represented by formula 3). As illustrated in Fig. (4B), at lower con-
centrations of the inhibitor significant differences in heparanase-
inhibition activities between heparin and N-acetyl heparin are
clearly observable. 2-O-desulfated derivatives were shown to retain
the heparanase inhibition activity of the parent heparin [92]. In fact,
neither 2-O-sulfate nor 6-O-sulfate groups are essential for efficient
inhibition of heparanase, provided that at least one of these posi-
tions is extensively sulfated [62]. Relationships between structure
and heparanase inhibition activity of heparin were established using
a number of heparins and heparin derivatives, including some with
various degrees of 6-O-sulfation of GlcN and 2-O-sulfation of IdoA
residues, as well as “glycol-split” derivatives obtained by controlled
periodate oxidation/borohydride reduction of natural [93] or par-
tially 2-O-desulfated heparins [94,95]. Glycol-splitting of C2-C3
bonds of nonsulfated uronic acid residues was suggested to modu-
late the biological interactions of heparin by providing flexible
joints between protein binding sequences [62, 94-98]. Notably,
when framing heparin sequences that bind FGF-2, glycol-split resi-
dues do not impair the binding to the growth factor; however, they
prevent activation of FGF-2 and FGF-2-induced angiogenic activity
[94,95].
Heparins with various degrees of N-acetylation/N-sulfation, to-
gether with some of their glycol-split derivatives, were investigated
for their FGF-2 and heparanase-inhibition activity [62]. Partially N-
acetylated heparins listed in Table 1 had different percentages of N-
acetyl groups, the complement to 100% N-substitution being N-SO3
groups. Glycol-split heparins and glycol-split N-acetyl heparins
were of two different types. The first type, referred to as “reduced
oxyheparins” (RO-H) and “N-acetyl-reduced oxyheparins” (NA-
RO-H), involved glycol-splitting of only the nonsulfated uronic
acid residues (GlcA and IdoA) originally present in the parent hepa-
rin [62]. De-O-sulfation of one out of two IdoA residues followed
by glycol-splitting gave heparins denoted H50gs with prevalent
Current Pharmaceutical Design, 2007, Vol. 13, No. 20 2065
structure 4, corresponding to sequences of poly-pentasulfated tri-
saccharides separated by glycol-split uronic acid residues [94].
The heparanase inhibitory activity (expressed as percent inhibi-
tion of heparanase) of heparin and heparin derivatives at concentra-
tion of 1 g/ml, is shown in Table 1 [62]. These data confirm that
heparin is a strong inhibitor of heparanase (~70% inhibition at 1
g/ml). Significant differences in heparanase-inhibiting activity
were associated with specific chemical modifications of heparin.
Whereas either 6-O-desulfation or 2-O-desulfation with retention of
L-IdoA configuration had little or no effect on the heparanase in-
hibitory activity of heparin, 2-O-desulfation with change of con-
figuration of the L-IdoA residues to L-GalA (involved in alkali
treatment of heparin under well defined conditions) [99]) markedly
decreased the inhibitory activity of heparin. Also, complete removal
of N-sulfate groups followed by N-acetylation resulted in a substan-
tial decrease in the inhibitory activity (Fig. 4B). However, this ef-
fect was only noted for N-acetylation degrees higher than approxi-
mately 50%. On the other hand, glycol-splitting markedly increased
the heparanase-inhibiting activity of both heparins and N-acetylated
heparins and restored the inhibitory effect lost upon N-acetylation
of heparin. This effect is illustrated by data in Table 1 for N-
acetylated heparins of the RO-type (i.e., 25% glycol-split), which
almost completely inhibited the heparanase activity (to less than
10% of the control at 1 g/ml and to 20-30% at 0.20-0.25 g/ml),
irrespective of their degree of N-acetylation. Glycol-splitting ex-
tended to newly-generated non-sulfated IdoA/GalA residues in
heparin and N-acetylated heparins yielded products all showing
high heparanase inhibitory activity without apparent dependence on
the degree of glycol splitting. The heparanase IC50 values calculated
from dose-dependence curves were >5 g/ml for NAH, ~0.4 g/ml
for H (heparin), and ~0.2 g/ml for 100NA, RO-H [62]. As shown
by data in Table 1 and illustrated in Fig. (4), 100NA,RO-H emerged
as an inhibitor of heparanase stronger than heparin. Data in Table 1
also show that it is consistently stronger than maltohexaose sulfate
(MHS, formula 8). Gel permeation chromatographic analysis of
some products of heparanase digestion, performed under conditions
of the enzyme inhibition assay, indicated that whereas heparin and
N-acetyl heparin are cleaved by heparanase (as previously shown
for heparin) [14], their glycol-split derivatives are not susceptible to
cleavage [62].
2066 Current Pharmaceutical Design, 2007, Vol. 13, No. 20 Casu et al.

Table 1. Distribution of Major Sulfate Groups and Heparanase Inhibitory Activity of Heparins and Derivatives [62]

Heparanase inhibitory activity, %

SO3 groups (%)
Mean [SD (N) ]a

NS A6OS I2OS 1 g/ml

HEPARINS

H1 89 79 69 73.5 [13.5 (7)]

6-O-DESULFATED H-1

H, 716OdeS (A) 81 29 55 50.5 [25.6 (7)]

77
H, 6Odes (B) 87 23 67 66.0 [ 9.4 (2)]
73
H, 6OdeS (B) 78 27 64 70.9 [18.0 (3 )]
46
H, 6OdeS (B) 82 56 68 63.4 [17.0 (6)]

2-O-DESULFATED H-1

H,IdoA (A) 83 85 0 64.7 [17.8 (3)]

H,GalA (B) 86 74 0 11.5 [11.0 (2)]

N-DESULFATED, N-ACETYLATED H-1

29
NAH 71 80 72 88.4 [11.6 (4)]
39
NAH 61 80 71 76.6 [7.4 (2)]
50
NAH 50 79 70 52.0 [ 4.4 (2)]
58
NAH 41 79 68 32.9 [15.3 (2)]
70
NAH 30 75 65 37.9 [20.2 (5)]
99
NAH 8 71 73 32.7 [18.0 (4)]
100
NAH 0 78 66 32.9 [15.3 (2)]

GLYCOL-SPLIT H-1

RO-H 89 75 67 91.3 [5.8 (2)]

52
H, gs 89 75 48 79.1 [17.7 (6)]

N-ACETYLATED, GLYCOL-SPLIT H-1

26
NA,RO-H 74 80 77 91.1 [3.1 (4)]
40
NA,RO-H 60 80 71
53
NA,RO-H 47 79 71 94.8 [3.1 (6)]
67
NA,RO-H 33 79 79 93.9 [2.2 (3)]
100
NA,RO-H 0 71 75 92.5 [5.0 (3)]
29 60
NAH, gs 71 75 40 79.6 [13.0 (2)]
43 60
NAH, gs 57 75 40 70.1 [17.2 (2)]
57 64
NAH, gs 42 75 36 87.0 [10.5 (2)]
70 59
NAH, gs 30 75 41 87.8 [12.5 (2)]

LMW HEPARINS and DERIVATIVES H-1

LMW-H-1 6.5KD 82 77 66 47.7 [35.0 (2)]

49
H, gs 6.3 KD 87 75 51 86.2 [ 2.8 (3)]
49
H, gs 3.0KD 89 75 51 [10.0 (2)]
50
LMW, NA,RO-H 5.4KD 50 79 75 90.4 [3.0 (3)]

MHS ~2.5 KD - - - 69.2 [5.7. (4)]

a 2
SD standard deviation = (
(yi-ymean) /(N-1)). N = number of experiments.
Heparanase Functions and Inhibition
4.3. Effect of Modified Heparins on Angiogenic Properties, Re-
lease of ECM-Bound FGF-2, and Stimulation of FGF-2 Mito-
genic Activity
Some species of heparin, and especially most of its glycol-split
derivatives that are potent inhibitors of heparanase, are endowed
with antiangiogenic properties also because they prevent activation
of growth factors such as FGF-2 [94, 95] and VEGF [100]; re-
viewed in [101]. A significant relationship was found between the
extent of glycol-splitting and the FGF2-antagonist/angiostatic ac-
tivities of glycol-split heparins [95]. However, comparison of struc-
ture-activity relationships for inhibition of growth factors and inhi-
bition of their release from HSPGs (via inhibition of heparanase)
underlines significant differences. Notably, whereas unmodified
heparin is a potent inhibitor of heparanase [62, 87], it is either inac-
tive or even pro-angiogenic in typical tests such as the CAM [94]
and several in vivo assays [R Giavazzi, C Pisano et al., unpub-
lished]. On the other hand, whereas glycol-split N-sulfated heparins
inhibit both growth factors and heparanase, their N-acetylated ana-
logs are inactive towards most growth factors but are as active as
their N-sulfated analogs in inhibiting heparanase. Inhibition of
growth factors and inhibition of heparanase show also different
dependences on the lenght of heparin sequences framed by glycol-
split residues [62,95, and unpublished results].
Some of the heparin derivatives were tested for their capacity to
release FGF-2 from ECM [62, 87]. As illustrated in Fig. (5), dose-
response curves of the FGF-2-releasing activity of glycol-split
heparin (H,52gs) and its corresponding low-molecular weight de-
rivative (LMW-H,49gs) were almost superimposable to those re-
ported for heparin [62], indicating that glycol-splitting does not
substantially modify the FGF-2-releasing properties of heparins.
Since glycol-splitting impairs oligomerization of FGF-2 [94, 95], it
is expected that glycol-split heparins release the growth factor
largely in an inactive form. Also, the curves of RO-H (i.e., heparin
glycol-split only at the level of pre-existing nonsulfated GlcA and
Fig. (5). Effect of combined N-acetylation and glycol-splitting on release
of ECM-bound FGF-2. ECM-coated wells were incubated (3 h, 24°C) with
iodinated FGF-2 and the unbound FGF-2 was washed away. The ECM was
then incubated (3 h, 24°C) with the indicated species of N-acetyl and glycol-
split heparins and aliquots of the incubation medium were counted in a
gamma counter. The remaining ECM was solubilized and its radioactivity
counted and used to calculate the percentage of ECM-bound 125I-FGF-2
released by each compound [62].
Current Pharmaceutical Design, 2007, Vol. 13, No. 20 2067
IdoA residues) are superimposable on that of the parent heparin
(data not shown). Data in Fig. (5) confirm that glycol-split, N- ace-
tylated heparins behave similarly to non glycol-split NAH [62] in
that they release ECM-bound FGF-2 consistently less than un-
modified heparin. 100NAH (not shown) and 100NA,RO.H exhibited
the lowest FGF-2 releasing activity among the tested compounds,
yielding only about twice the spontaneous release observed in pres-
ence of the buffer (PBS) alone.
The ability of heparin, 100NAH and 100NA,RO.H to promote the
mitogenic activity of recombinant FGF-2 was investigated using a
cytokine-dependent, heparan sulfate deficient, lymphoid cells
(BaF3) engineered to express FGFR1. Unlike heparin, both fully N-
acetylated heparin (100NAH) and its glycol-split counterpart mole-
cule (100NA,RO.H) failed to stimulate the mitogenic activity of
FGF-2, beyond the basal level obtained in the absence of the added
heparin (Fig. 6). Thus, while glycol splitting of NAH fully restores
its heparanase-inhibiting activity, it fails to induce a similar restora-
tion of the ability to displace ECM-bound FGF-2 and to stimulate
the mitogenic activity of recombinant FGF-2 [62].
Fig. (6A). Idealized representation of stimulation by heparin of FGF-2-
induced lymphoid cell proliferation. B) Effect of N-acetylation and glycol-
splitting on stimulation of the mitogenic activity of FGF-2. BaF3 (F32)
lymphoid cells were plated into 69-well microtiter plates in the presence of
2.5 ng/ml FGF-2 and increasing concentrations of heparin /H), totally N-
acetylated heparin (NAH), or the corresponding glycol-split derivtive of
RO-type. After 48 h, 3H-thymidine was added (1 Ci/well) and the amount
of incorporated tymidine was determined as described in Ref. [67].
2068 Current Pharmaceutical Design, 2007, Vol. 13, No. 20
5. MODELING CONSIDERATIONS
Determination of the molecular conformation of HS/heparin
chains recognized and cleaved by heparanase, as well as those that
effectively inhibit its enzymatic activity, is a formidable task. Even
modeling based on established criteria of conformational energies
must take into account the extraordinary conformational versatility
of GAG chains that contain IdoA residues. IdoA residues have been
demonstrated to be endowed with a unique conformational flexibil-
ity (“plasticity”). Such a plasticity, associated with different
equienergetic conformations of IdoA residues, all co-existing in a
rapid dynamic equilibrium, can currently explain the better protein
binding capacity and associated biological properties of IdoA-
containing sequences, as compared to the more rigid GlcA-
containing ones [89, 102]. As demostrated for complexes with other
proteins such as antithrombin both in crystals [103] and in solution
[104] and for FGF-2 in crystals [105], heparin oligosaccharides
may select one of the most favored conformations of its IdoA (or
IdoA2SO3) residues for optimal docking to the protein [89,106]. It
is thus possible that some of the iduronate residues along the same
heparin (or heparin derivative) chain adopt different local confor-
mations depending on the 3D structure of the protein to which it
binds.
Although fine details of the 3D structure of heparanase are not
yet known, examination of models thus far available permits to
envisage how the enzyme binds to and cleaves the HS chains of
HSPGs. As illustrated in Fig. (7), an homology model of hepara-
nase using the X-ray structure of 1,4-
-xylanase [39] suggests that
HS chains can be accommodated in a canyon in the active form of
the protein [26]. The glycosidic linkage of the GlcA residue suscep-
tible to cleavage by heparanase is expected to be docked to the
active site of the enzyme, which is approximately located at the
center of the canyon. The oligosaccharide sequences from both
sides of this GlcA residue conceivably bind from one side to HBD-
1 and from the other side to HBD-2. Since the distance between
HBD-1 and HBD-2 is of the order of an octa-decasaccharide [M.
Guerrini, personal communication], the optimal length of the HS
Fig. (7). Homology modeling of heparanase using the X-ray structure of
1,4-
-xylanase as a template [39].The yellow letters represent the conserved
critical residues of Glu225 (proton donor) and Glu343 (nucleophile), respec-
tively. The HS chain was manually located on the active site of heparanase,
and the complex model was constructed using molecular mechanics and
molecular dynamics calculations with consideration of the mechanism of
enzymatic glycoside hydrolysis [26].
Casu et al.
sequences for binding to both heparin/HS binding domains 1 and 2
should correspond to an octa- decasaccharide, including the GlcA
residue. Heparin tetra and hexasaccharides are poor inhibitors of
heparanase [87, and our own unpublished data]. Most probably,
these short non-GlcA-containing oligosaccharides bind to either
HBD-1 or HBD-2 (or, at high concentrations, to both), but such a
binding is weak when it is not strenghtened by any interaction with
the active site of the enzyme. On the other hand, small heparin
oligo-saccharide chains terminating with an aza-sugar (exemplified
by formula 7, where R may be another heparin oligosaccharide)
effectively inhibit heparanase [107], suggesting that combination of
blocking the active site of the enzyme with the aza sugar residue
and either HBD-1 or HBD-2 (or both basic domains) with heparin
oligosaccharide sequences, is effective in preventing recognition
and cleavage of HS chains by heparanase.
Full length heparins are strong inhibitors of heparanase in spite
of being also substrates for the enzyme [14]. However, not all the
SO3 groups of heparin are essential for the inhibition of the enzy-
matic activity. Such a behavior is compatible with an accepted
model of heparin chains where clusters of three sulfate groups al-
ternate on both sides of a helix, irrespective of the conformation of
iduronate residues [108] (Fig. 8a). The assumption that only sulfate
groups on one side of the helix are involved in the interaction with
basic clusters of heparanase is compatible with the observation that
heparins whose N-SO3 groups have been replaced up to 50% by N-
acetyl groups retain the heparanase-inhibition activity of the parent
heparin [62,98]. On the other hand, complete removal of either 6-O-
sulfate or 2-O-sulfate groups does not significantly impair the
heparanase inhibition activity of heparin, provided the GAG is fully
N-sulfated and 2-O- (or 6-O)- sulfated, respectively [62].
The impressive effectiveness of glycol-split heparins as hepara-
nase inhibitors raises the question of the role played by glycol-split
residues in boosting such an activity. It was predicted [96] and ex-
perimentally proved [62,109] that glycol-splitting provides extra-
flexibility to heparin (GAG) chains while not intefering with the
binding properties of chains segments that do not contain glycol-
split residues. As mentioned before and illustrated in Fig. (8a),
short segments of N-sulfated heparin chains can be represented as
linearly propagating helices featuring arrays of NSO3, 2SO3, and
6SO3 groups alternating on each side of the chain [108]. Because of
limited rotation allowed around ”normal” glycosidic bonds, these
arrays cannot align themselves on the same side of the heparin
chain. On the contrary, glycol-splitting introduces extra degrees of
rotational freedom and may generate novel conformations such as
those shown in Fig. (8b) and (8c). Conformation b, which is one of
the two energetically favored conformations compatible with NMR
data, features a “kink” at the level of the glycol-split residue; a sec-
ond permitted conformation compatible with experimental data,
also showing the kink, is symmetrical to the one in b [95]. It must
be noted that glycol-split heparins may also adopt conformations
such as that shown in Fig. (8c), with arrays of up to four sulfate
groups in a row. At least in principle, such conformations may be-
came favored by complexing with heparanase. More refined model-
ing studies are needed to elucidate the actual conformation adopted
by HS/heparin chains both in the absence and in the presence of
heparanase. Further studies are also needed to rationalize the nota-
ble finding that whereas replacement with N-acetyl groups of all the
N-sulfate groups of heparin impairs the heparanase inhibition activ-
ity, glycol-splitting restores this activity to the level of the corre-
sponding glycol-split, N-sulfated heparins [62]. It is most probable
that glycol-split N-acetylated heparins adopt, in their complexes
with heparanase, a conformation that is different from that of N-
sulfated heparins, conceivably also because of requirements for
some of their N-acetyl groups to settle in hydrophobic niches of the
protein. Molecular modeling studies indicate that the overall con-
formation of N-acetyl heparin (Fig. 8d) does not substantially differ
from that of N-sulfated heparin (Fig. 8a) [110]. Also the favored
“kinked” conformations of glycol-split N-acetyl heparin (such as
Heparanase Functions and Inhibition Current Pharmaceutical Design, 2007, Vol. 13, No. 20 2069

that represented in Fig. 8e) do not substantially differ from the cor-
responding ones of the glycol-split N-sulfated heparin compatible
with experimental NOE values [98]. Both N-acetyl heparin in its
energetically favored conformation 8d [110] and in its “kinked”
form (8e) feature only two sulfate groups (as opposed to three for
N-sulfated heparin) regularly alternating on both sides of the GAG
chains. However, similarly to glycol-split N-sulfated heparins, in
some of the energetically allowed conformations of glycol-split N-
acetyl heparins such as that shown in Fig. (8f), arrays of sulfate
groups of adjacent disaccharide units may be brought close to each
other on the same side of the GAG chain [S. Guglieri et al., unpub-
lished results]. Heparin/HS chains in these conformations may be
stabilized and selected by heparanase to form stable complexes.
Thus, the combination of local flexibility associated with the intrin-
sic plasticity of sulfated iduronate residues and the extra-flexibility
induced by glycol-splitting seems to favor optimal “exploitation” of
sulfate groups in binding to basic sites lying along the canyon of
heparanase and lead to effective inhibition of the enzyme by hepa-
rin derivatives having not more than two sulfate groups per disac-
charide unit.
Although not all the sulfate groups of a extensively sulfated
polysaccharide are necessarily involved in protein binding [3],
some persulfated poly- and oligosaccharides are often stronger
binders than the corresponding less sulfated species [86, and our
own unpublished data]. Data in Table 1 indicate that the hexa-
saccharide MHS (which, as shown in formula 8, may bear up to
three sulfate groups per each internal disaccharide residue and up to
four for the terminal ones), though less potent than glycol-split
heparins, is also a good inhibitor of heparanase [86]. Carbohydrate
backbones such as those generated by the
-1,4-linked glucose
residues of MHS are characterized by some flexibility associated
with rotation of residues around the axial glycosidic bonds. It is
accordingly expected that the MHS molecules (Formula 8) adjust
themselves in the heparanase molecule in such a way that most of
the basic residues of sites HBD1 and HBD2 are blocked by the
sulfated oligosaccharide. The recently reported heparanase inhibitor
oligomannurarate sulfate (JG3) is suggested to bind to HBD1
[111].
6. CONCLUSIONS
Although a significant progress has been made during the last
years in understanding heparanase biology, there is much to be
learned. Accumulation of compelling evidence implies that the
enzyme is up regulated in primary human tumors and inversely
correlates with survival rate of cancer patients post operation. An-
giogenesis is the primary 'suspect' that governs heparanase medi-
ated tumor progression. This mode of action and the related clinical
applications await further confirmation and require new molecular
tools such as small inhibitory molecules, neutralizing antibodies,
and heparanase-inhibiting glycol-split species of heparin with in
vivo pharmacological and pharmacokinetics tailored to the specific
therapeutic application, as well as an effective approach to deliver
anti-heparanase agents into the tumor tissue. The ability of hepara-
nase to function in an apparently enzymatic independent manner,
noted in several experimental settings [55, 65, 70, 72, 73], is in-
triguing and affects the way the protein is envisioned. Thus, while
attention was mainly focused on compounds that inhibit heparanase
enzymatic activity, no information is available on protein domains
responsible for the non-enzymatic functions of the heparanase en-
zyme. In this respect, identification of a putative heparanase recep-

Fig. (8). Minimized structure of heparin octasaccharide sequences. (a) heparin; (b) and (c), glycol-split heparin in its kinked and extended form, respec-
tively; (d) N-acetylated heparin; (e) and (f), glycol-split N-acetylated heparin in its kinked and extended form, respectively. (See text). Model a) was con-
structed using pdb code 1HPN [108], with IdoA2SO3 residues in the 1C4 conformation. Minimized structures b-f were obtained by modifying a). Arrows indi-
cate IdoA2SO3 residues that are 2-O-desulfated and glycol-split in strucures b-f. Glycol-split residues are circled.
2070 Current Pharmaceutical Design, 2007, Vol. 13, No. 20 Casu et al.

tor is a major future challenge. Some of the N-acetylated glycol- GPI-anchored = Glycosyl phosphatidyl inositol-anchored
split heparins (such as 100NA, ROH) emerge as promising hepara- TNF
= Tissue necrosis factor

nase-inhibiting lead compounds. Another important objective is the
establishment of a reliable diagnostic assay to monitor heparanase PKC = Protein kinase C
levels and activity in plasma and urine of cancer patients by mean PKA = Protein kinase A
of a sensitive, high throughput activity assay and ELISA method. HDB = Heparin binding domain
The secreted nature of the enzyme and its induction in primary
RLP = Lipoprotein receptor-related protein
human tumors predict that under certain conditions the protein is
present in body fluids. It is conceivable that elevated levels of RT-PCR = Reverse transcriptase polymerase chain
heparanase are found in the plasma and/or urine of cancer patients reaction
as well as in other pathological disorders such as diabetes [112- MRI = Magnetic resonance imaging
114]. Induction of heparanase already at early phases of cancer SMA = Smooth muscle actin
progression supports an important diagnostic and, possibly, prog-
nostic value of such assays. HB-EFG = Heparin-binding epidermal growth factor
Recent publications clearly imply that heparanase may be in- KGF = Keratinocyte growth factor
volved in pathological conditions other than cancer. An intriguing Hpa-tg = Heparanase transgenic mice
example is the observation that heparanase over expressing trans- PECAM-1 = Pletelet endothelial cell adhesion molecule-1
genic mice escaped amyloid deposition in experimental models of
inflammatory-associated amyloidosis [115] and prion diseases (our HBP = Heparin binding protein
unpublished observations). The possible involvement of heparanase Cyto.D = Cytochalasin D
in degenerative diseases such as Alzheimer's disease is only starting MRS = 2'-Deoxy-N6-methyladenosine-3',5'-
to emerge and should be perused, taking into account the increasing bisphosphate
significance of these illnesses. Likewise, the causal involvement of
PPADS = Pyridoxal-phosphate-6-azophenyl-2'-
heparanase in diabetic nephropathy [116], auto-immunity [117] and
4'disulphonic acid
inflammatory disorders [85] should be investigated.
siRNA = Small interference RNA
Functional domains other than the basic heterodimer structure
[35-37] and amino acids (Glu225, Glu343) critical for the enzyme DTH = Delayed type hypersensitivity
catalytic activity [22], have not been so far identified in the hepara- GlcA = Glucuronic acid
nase protein, making screens for inhibitory molecules random in MHS = Maltohexaise sulfate
nature. The identification of heparin binding domains [56] and the
ability of the corresponding KKDC peptide, especially when ap- PI-88 = Phosphomannopentaose sulfate
plied as a dimer, to inhibit heparanase enzymatic activity [56], GAG = Glycosaminoglycan
clearly emphasize the need for crystallization and accurate under- AT = Antithrombin
standing of the 3D structure of the enzyme toward an efficient drug
development program. While it is now well accepted that a single GFR = Growth factors
active heparanase enzyme is expressed by mammals, heparanase NAH = N-acetyl heparin
splice variants have recently been characterized in the Mole rat NA-ROH = N-acetyl reduced oxyheparin
[118] and human (our unpublished results), although their role has
IdoA = Iduronic acid
not been established. The heparanase system may be envisaged to
include heparanase 1 and its splice variants, the three splice variants GalA = Galacturonic acid
of heparanase 2 [119], the heparanase processing protease, and, CAM = Chicken chorioallantoic membrane
possibly, the heparanase cell surface receptor. Gain of more infor- LMW-H = Low-molecular weight heparin
mation will enable the development of new inhibitory strategies
directed against the enzymatic and non-enzymatic functions of RO.H = Reduced oxyheparin
heparanase, altogether offering better therapeutic opportunities. FGFR1 = Fibroblast growth factor receptor 1
NOE = Nuclear Overhauser effect.
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